Many plants are genetically transformed with genes from other species to introduce desirable traits, such as to improve agricultural value through, e.g., improving nutritional value quality, increasing yield, conferring pest or disease resistance, increasing drought and stress tolerance, improving horticultural qualities such as pigmentation and growth, and/or imparting herbicide resistance; enabling the production of industrially useful compounds and/or materials from the plant; and/or enabling the production of pharmaceuticals. The introduction of cloned genes into plant cells and recovery of stable fertile transgenic plants can be used to make such modifications of a plant, and has allowed desirable traits or qualities of interest to be incorporated into plants via genetic engineering (e.g., crop improvement). In these methods, foreign DNA is typically introduced into the nuclear or plastid DNA of the eukaryotic plant cell, followed by isolation of cells containing the foreign DNA integrated into the cell's DNA, to produce stably transformed plant cells.
One drawback that arises regarding the use of transgenic plants is the possibility of transgene escape to wild species and non-transformed species. These traits can increase the risk of outcrossing, persistence, and introgression of transgenes into an adjacent population. The escape of transgenes from genetically modified (GM) crops usually occurs through gene flow, mainly by cross-pollination (Lu (2003) Eviron. Biosafety Res. 2:3-8), but may also occur through introgression. Stewart Jr. et al. (2003) Nat. Reviews Gen. 4:806-17. Crop-to-crop gene flow will result in contamination of non-GM varieties, affecting the strategic deployment of transgenic and non-transgenic crop varieties in a given agricultural system. Significant contamination of non-GM crops with transgenic material poses difficulties in international trade because of legal restrictions on imports of transgenic products by many countries. Crop-to-crop gene flow can cause stacking of transgenes in hybrids that may potentially become volunteer weeds if the transgenes impart multiple resistance (e.g., to herbicides, pests, and/or diseases). Additionally, crop-to-crop gene flow will lead to transgene escape into weedy populations or related wild species, which may pose serious weed problems and other ecological risks if the transgenes persist and establish in the weedy/wild populations through sexual reproduction and/or vegetative propagation. This is a particular concern when escaped genes enhance the ecological fitness of the weedy/wild species. Introgression of a crop transgene occurs in steps involving several successive hybrid generations. Introgression is a dynamic process that may take many years and generations before the transgene is fixed in the genetic background of a receiving species and, thus, presents difficulties of detection and monitoring. However, if selection is strong and/or population size is small, fixation of an introgressed gene may occur rapidly.
Containment of a specific expression cassette within genetically modified plants, especially a selectable marker expression cassette, is an elusive goal. Selectable marker genes are usually antibiotic resistant or herbicide tolerant genes, but may include reporter genes (i.e., β-glucuronidase (Graham et al. (1989) Plant Cell Tiss. Org. 20(1):35-39). Selectable makers which are co-transferred into the genome of a plant provide a selective advantage and allow for the identification of stably transformed transgenic plants. The availability of functional selectable maker genes which can be used for the transformation of plants is somewhat limited. A review of the published scientific literature on transgenic crop plants reveals that the most widely used selective agents for antibiotic resistance are for kanamycin (encoded by the neomycin phosphotransferase type II gene (Bevan et al. (1983) Nature 304:184-187)) or hygromycin (encoded by the hygromycin phosphotransferase gene (Waldron et al., Plant Mol. Biol. 5:103-108)), and herbicide tolerance is phosphinothricin resistance (encoded by the pat (Wohlleben et al. (1988) Gene 70:25-37) or bar genes (DeBlock et al. (1987), EMBO J. 6 (9):2513-2518)). See, Sundar et al. (2008) J. Plant Physiol. 165:1698-1716. Given the limited number of selectable marker genes and the common use of a sub-set of these traits, a solution that allows for the excision and re-use of selectable markers within a transgenic plant would obviate the need for additional selectable makers in subsequent rounds of gene transfer or gene stacking into the same plant. Moreover, the ability to excise a selectable marker could overcome unintended changes to the plant transcriptome that are caused by the expression of the marker (Abdeen et al. (2009) Plant Biotechnol. J. 7(3):211-218).
Current strategies to prevent or minimize gene flow between GM crops and other species and varieties include: (1) physical isolation of the transgenic crop; (2) chloroplast engineering of transgenes; (3) co-engineering of a mitigation gene along with the transgene; (4) genetic use restriction technologies (GURTs); (5) CRE/loxP and FLP/FRT recombinase-mediated gene deletion. See, e.g., Lee and Natesan (2006) TRENDS Biotech. 24(3):109-14; Lu (2003), supra; and Luo et al. (2007), Plant Biotech. J. 5:263-74; and (6) meganuclease-mediated gene deletion. See, e.g., U.S. patent application Ser. No. 11/910,515; and U.S. patent application Ser. No. 12/600,902.
CRE, FLP, and R recombinases have been exploited for the excision of unwanted genetic material from plants. Hare and Chua (2002) Nat. Biotech. 20:575-80. Luo et al. (2007), supra, reported a pollen- and seed-specific “GM-gene-deletor” system, wherein use of loxP-FRT fusion sequences as recognition sites for excision of transgenes by CRE or FLP recombinase led to deletion of transgenes from pollen, or from both pollen and seed, of transgenic tobacco plants. All these site-specific recombinase systems shown to function in plants are members of the integrase family. These systems have been chosen for use, at least in part, due to the fact that other recombinases may require ancillary proteins and more complex recognition sites that may confer topological restraints on recombination efficiencies. Id. These systems have several significant drawbacks: integrase-type recombinases may also recognize “pseudo-sequences,” which may be highly divergent from a specific target sequence and, therefore, lead to unwanted non-specific DNA deletions; and excision of a target sequence leaves a residual recognition sequence that may be sites of chromosomal rearrangements upon subsequent exposure to the recombinase, or activate gene silencing mechanisms. Id. Moreover, these systems are further constrained as a functional recombinase must be present and expressed in one of the parent plants, the presence of which requires additional strategies for deletion within pollen and/or seed. Despite these limitations, the CRE/loxP system is recognized as the most suitable strategy for optimization of gene deletion in plants. Id.
Custom-designed zinc finger nucleases (ZFNs) are proteins designed to deliver a targeted site-specific double-strand break in DNA, with subsequent recombination of the cleaved ends. ZFNs combine the non-specific cleavage domain of FokI restriction endonuclease with zinc finger DNA-binding proteins. See, e.g., Huang et al. (1996) J. Protein Chem. 15:481-9; Kim et al. (1997) Proc. Natl. Acad. Sci. USA 94:3616-20; Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93:1156-60; Kim et al. (1994) Proc. Natl. Acad. Sci. USA 91:883-7; Kim et al. (1997b) Proc. Natl. Acad. Sci. USA 94:12875-9; Kim et al. (1997c) Gene 203:43-9; Kim et al. (1998) Biol. Chem. 379:489-95; Nahon and Raveh (1998) Nucleic Acids Res. 26:1233-9; Smith et al. (1999) Nucleic Acids Res. 27:674-81. Individual zinc finger motifs can be designed to target and bind to a large range of DNA sites. Cys2His2 zinc finger proteins bind DNA by inserting an α-helix into the major groove of the double helix. Recognition of DNA by zinc fingers is modular: each finger contacts primarily three consecutive base pairs in the target, and a few key residues in the protein mediate recognition. It has been shown that FokI restriction endonuclease must dimerize via the nuclease domain in order to cleave DNA, inducing a double-strand break. Similarly, ZFNs also require dimerization of the nuclease domain in order to cut DNA. Mani et al. (2005) Biochem. Biophys. Res. Commun. 334:1191-7; Smith et al. (2000) Nucleic Acids Res. 28:3361-9. Dimerization of the ZFN is facilitated by two adjacent, oppositely oriented binding sites. Id. In addition, double strand breaks caused by zinc finger nucleases are resolved by the plants DNA repair machinery via either nonhomologous end joining (NHEJ) or homology directed repair (HDR), thereby resulting in plants which are free of residual recognition sequences.